4-aminopyridine improves evoked potentials and ambulation in the taiep rat: A model of hypomyelination with atrophy of basal ganglia and cerebellum.

The taiep rat is a tubulin mutant with an early hypomyelination followed by progressive demyelination of the central nervous system due to a point mutation in the Tubb4a gene. It shows clinical, radiological, and pathological signs like those of the human leukodystrophy hypomyelination with atrophy of the basal ganglia and cerebellum (H-ABC). Taiep rats had tremor, ataxia, immobility episodes, epilepsy, and paralysis; the acronym of these signs given the name to this autosomal recessive trait. The aim of this study was to analyze the characteristics of somatosensory evoked potentials (SSEPs) and motor evoked potentials (MEPs) in adult taiep rats and in a patient suffering from H-ABC. Additionally, we evaluated the effects of 4-aminopyridine (4-AP) on sensory responses and locomotion and finally, we compared myelin loss in the spinal cord of adult taiep and wild type (WT) rats using immunostaining. Our results showed delayed SSEPs in the upper and the absence of them in the lower extremities in a human patient. In taiep rats SSEPs had a delayed second negative evoked responses and were more susceptible to delayed responses with iterative stimulation with respect to WT. MEPs were produced by bipolar stimulation of the primary motor cortex generating a direct wave in WT rats followed by several indirect waves, but taiep rats had fused MEPs. Importantly, taiep SSEPs improved after systemic administration of 4-AP, a potassium channel blocker, and this drug induced an increase in the horizontal displacement measured in a novelty-induced locomotor test. In taiep subjects have a significant decrease in the immunostaining of myelin in the anterior and ventral funiculi of the lumbar spinal cord with respect to WT rats. In conclusion, evoked potentials are useful to evaluate myelin alterations in a leukodystrophy, which improved after systemic administration of 4-AP. Our results have a translational value because our findings have implications in future medical trials for H-ABC patients or with other leukodystrophies.

CRK12: A Key Player in Regulating the Phaseolus vulgaris-Rhizobium tropici Symbiotic Interaction.

Cysteine-rich receptor-like kinases (CRKs) are a type of receptor-like kinases (RLKs) that are important for pathogen resistance, extracellular reactive oxygen species (ROS) signaling, and programmed cell death in plants. In a previous study, we identified 46 CRK family members in the Phaseolus vulgaris genome and found that CRK12 was highly upregulated under root nodule symbiotic conditions. To better understand the role of CRK12 in the Phaseolus-Rhizobia symbiotic interaction, we functionally characterized this gene by overexpressing (CRK12-OE) and silencing (CRK12-RNAi) it in a P. vulgaris hairy root system. We found that the constitutive expression of CRK12 led to an increase in root hair length and the expression of root hair regulatory genes, while silencing the gene had the opposite effect. During symbiosis, CRK12-RNAi resulted in a significant reduction in nodule numbers, while CRK12-OE roots showed a dramatic increase in rhizobial infection threads and the number of nodules. Nodule cross sections revealed that silenced nodules had very few infected cells, while CRK12-OE nodules had enlarged infected cells, whose numbers had increased compared to controls. As expected, CRK12-RNAi negatively affected nitrogen fixation, while CRK12-OE nodules fixed 1.5 times more nitrogen than controls. Expression levels of genes involved in symbiosis and ROS signaling, as well as nitrogen export genes, supported the nodule phenotypes. Moreover, nodule senescence was prolonged in CRK12-overexpressing roots. Subcellular localization assays showed that the PvCRK12 protein localized to the plasma membrane, and the spatiotemporal expression patterns of the CRK12-promoter::GUS-GFP analysis revealed a symbiosis-specific expression of CRK12 during the early stages of rhizobial infection and in the development of nodules. Our findings suggest that CRK12, a membrane RLK, is a novel regulator of Phaseolus vulgaris-Rhizobium tropici symbiosis.

Label-Free Non-Linear Optics for the Study of Tubulin-Dependent Defects in Central Myelin.

The satisfactory visualization of cytoskeletal components in the brain is challenging. The ubiquitous distribution of the networks of microtubules, microfilaments, and intermediate filaments in all the neural tissues, together with the variability in the outcomes of fluorescent protein fusion strategies and their limited applicability to dynamic studies of antibodies and drugs as chromophore vehicles, make classical optical approaches not as effective as for other proteins. When tubulin needs to be studied, the label-free generation of second harmonics is a very suitable option due to the non-centrosymmetric organization of the molecule. This technique, when conjugated to microscopy, can qualitatively describe the volumetric distribution of parallel bundles of microtubules in biological samples, with the additional advantage of working with fresh tissues that are unfixed and unpermeabilized. This work describes how to image tubulin with a commercial second harmonic generation microscopy setup to highlight microtubules in the tubulin-enriched structures of the oligodendrocytes, as in hypomyelination with atrophy of the basal ganglia and cerebellum (H-ABC) tubulinopathy, a recently described myelin disorder.

Concurrent optical inspection to boost characterization of plastic cortical bone under mechanical deformation.

The simultaneous surface and internal measurements from a chemically modified cortical bovine bone suffering a plastic range deformation are presented. Since the bone is an anisotropic structure, its mechanical response could be modified if its organic or inorganic phases change. The latter could result in high plastic deformations, where the interferometrical signal from an optical analysis is easily de-correlated. In this work, digital holography interferometry (DHI) and Fourier domain optical coherence tomography (FD-OCT) are used to analyze the plastic range deformation of the bone under compression. The simultaneous use of these two optical methods gives information even when one of them de-correlates. The surface results retrieved with DHI show the high anisotropy of the bone as a continuously increasing displacement field map. Meanwhile, the internal information obtained with FD-OCT records larger deformations at different depths. Due to the optical phase, it is possible to complement the measurements of these two methods during the plastic deformation.

H-ABC tubulinopathy revealed by label-free second harmonic generation microscopy.

Hypomyelination with atrophy of the basal ganglia and cerebellum is a recently described tubulinopathy caused by a mutation in the tubulin beta 4a isoform, expressed in oligodendrocytes. The taiep rat is the only spontaneous tubulin beta 4a mutant available for the study of this pathology. We aimed to identify the effects of the tubulin mutation on freshly collected, unstained samples of the central white matter of taiep rats using second harmonic generation microscopy. Cytoskeletal differences between the central white matter of taiep rats and control animals were found. Nonlinear emissions from the processes and somata of oligodendrocytes in tubulin beta 4a mutant rats were consistently detected, in the shape of elongated structures and cell-like bodies, which were never detected in the controls. This signal represents the second harmonic trademark of the disease. The tissue was also fluorescently labeled and analyzed to corroborate the origin of the nonlinear signal. Besides enabling the description of structural and molecular aspects of H-ABC, our data open the door to the diagnostic use of nonlinear optics in the study of neurodegenerative diseases, with the additional advantage of a label-free approach that preserves tissue morphology and vitality.

Surface Profile Studies of Photoinduced Gratings Made with DCG Films with Optional Papain Development.

The use of surface relief structures is increasing in the field of optics. A study of photoinduced relief using dichromated gelatin films with different thickness is described in this paper. Two light sources were used: a laser (λ = 468 nm) and an ultraviolet mercury-metal halide lamp. Gratings with low spatial frequencies were contact-copied on the DCG (dichromated gelatin) films. Two development processes were used, one included washing the plates with just water and the other with a mixture of water and papain. This enzyme is used to improve the gratings' relief which was studied with a profilometer. For the development process with just water, it was found that when gratings were recorded using visible or UV light, the height profile inversely correlated to spatial frequencies. For short exposure times, the reliefs showed a sinusoidal profile. When visible light was used, the DCG areas where the Ronchi grating had transparent slits showed a flat relief and the areas where the Ronchi grating had opaque slits showed a round peak, with the peak being taller than the flat surface. In contrast, when UV light was used, the flat surfaces were taller than the peaks. The relief height increased up to seven times when papain was used.

Longitudinal Evaluation of Cerebellar Signs of H-ABC Tubulinopathy in a Patient and in the taiep Model.

Hypomyelination with atrophy of the basal ganglia and cerebellum (H-ABC) is a central neurodegenerative disease due to mutations in the tubulin beta-4A (TUBB4A) gene, characterized by motor development delay, abnormal movements, ataxia, spasticity, dysarthria, and cognitive deficits. Diagnosis is made by integrating clinical data and radiological signs. Differences in MRIs have been reported in patients that carry the same mutation; however, a quantitative study has not been performed so far. Our study aimed to provide a longitudinal analysis of the changes in the cerebellum (Cb), corpus callosum (CC), ventricular system, and striatum in a patient suffering from H-ABC and in the taiep rat. We correlated the MRI signs of the patient with the results of immunofluorescence, gait analysis, segmentation of cerebellum, CC, and ventricular system, performed in the taiep rat. We found that cerebellar and callosal changes, suggesting a potential hypomyelination, worsened with age, in concomitance with the emergence of ataxic gait. We also observed a progressive lateral ventriculomegaly in both patient and taiep, possibly secondary to the atrophy of the white matter. These white matter changes are progressive and can be involved in the clinical deterioration. Hypomyelination with atrophy of the basal ganglia and cerebellum (H-ABC) gives rise to a spectrum of clinical signs whose pathophysiology still needs to be understood.

Auditory impairment in H-ABC tubulinopathy.

Hypomyelination with atrophy of the basal ganglia and cerebellum (H-ABC) is a neurodegenerative disease due to mutations in TUBB4A. Patients suffer from extrapyramidal movements, spasticity, ataxia, and cognitive deficits. Magnetic resonance imaging features are hypomyelination and atrophy of the striatum and cerebellum. A correlation between the mutations and their cellular, tissue and organic effects is largely missing. The effects of these mutations on sensory functions have not been described so far. We have previously reported a rat carrying a TUBB4A (A302T) mutation and sharing most of the clinical and radiological signs with H-ABC patients. Here, for the first time, we did a comparative study of the hearing function in an H-ABC patient and in this mutant model. By analyzing hearing function, we found that there are no significant differences in the auditory brainstem response (ABR) thresholds between mutant rats and WT controls. Nevertheless, ABRs show longer latencies in central waves (II-IV) that in some cases disappear when compared to WT. The patient also shows abnormal AEPs presenting only Waves I and II. Distortion product of otoacoustic emissions and immunohistochemistry in the rat show that the peripheral hearing function and morphology of the organ of Corti are normal. We conclude that the tubulin mutation severely impairs the central hearing pathway most probably by progressive central white matter degeneration. Hearing function might be affected in a significant fraction of patients with H-ABC; therefore, screening for auditory function should be done on patients with tubulinopathies to evaluate hearing support therapies.

Resultados del primer registro de implante de cardiodesfibriladores y resincronizadores SAC (RENCARE) / Results of the First SAC Implantable Cardioverter-Defibrillator and Cardiac Resynchronization Therapy Registry (RENCARE)

RESUMEN Introducción: La prevención de la muerte súbita y el tratamiento de la insuficiencia cardíaca son temas de gran importancia. Para prevenir la muerte súbita y mejorar el pronóstico de la insuficiencia cardíaca se utilizan los cardiodesfibriladores y cardioresincronizadores. Objetivos: Evaluar la cantidad y tipo de dispositivos implantados en nuestro país, así como las características de los pacientes, las complicaciones agudas y las que se presentan en el seguimiento. Material y métodos: Se realizó un estudio observacional, prospectivo, multicéntrico en centros de salud con la capacidad de implantar cardiodesfibriladores y cardioresincronizadores. Se incluyeron pacientes a los que se les realizó implante de estos dispositivos desde enero del 2016 hasta enero de 2017, con un seguimiento de 12 meses. Resultados: Se incluyeron 249 pacientes (edad promedio de 64,8 ± 13,7 años, 73,9% de sexo masculino, 72,1% con Fey < 35%). La etiología subyacente de la miocardiopatía era isquémica en el 39,8%, dilatada 26,7% y chagásica en el 11,2% de los casos. El 58% de los implantes realizados fueron cardiodesfibriladores y el 39%, cardiodesfibriladores asociados con cardioresincronizadores. El 84% de los procedimientos fueron primoimplantes. La indicación más frecuente del implante fue por prevención primaria de muerte súbita (67,9%). La tasa de complicaciones menores fue del 4,4% y no se reportaron complicaciones mayores. Conclusiones: El siguiente registro evidenció una gran proporción de implantes en pacientes con cardiopatía isquémica, la indicación principal fue por prevención primaria de muerte súbita y la tasa de complicaciones fue similar a la reportada internacionalmente.

ABSTRACT Background: Prevention of sudden death and treatment of heart failure are very important topics. Implantable cardioverter-defibrillator and cardiac resynchronization devices are used to prevent sudden death and improve heart failure symptoms and prognosis. Objectives: The aim of this study was to evaluate the number, type of implanted devices, clinical characteristics of the patients and acute and follow-up complications. Methods: An observational, prospective, multicenter study was carried out in healthcare centers with the capacity to implant cardioverter-defibrillator and cardiac resynchronization devices. The study included all patients who underwent implantation of these devices from January 2016 to January 2017, with a 12-month follow-up. Results: A total of 249 patients (73.9% men) with mean age of 64.8±13.7 years, and 72.1% with ejection fraction <35%, were included in the study. The underlying cardiomyopathy etiology was ischemic in 39.8% of cases, dilated in 26.7% and chagasic in 11.2%. Fifty-eight percent of implants were implantable cardioverter-defibrillators and 39% were cardioverter-defibrillators associated with cardiac resynchronization devices. In 84% of cases, procedures were first implants. The most frequent indica-tion of implantation was for primary prevention of sudden death (67.9%). Minor complication rate was 4.4% and no major complications were reported. Conclusions: The present registry evidenced a large proportion of cardioverter-defibrillator and cardiac resynchronization implants in patients with ischemic heart disease. The main indication was for primary prevention of sudden death and the complication rate was similar to that reported internationally.

MRI Features in a Rat Model of H-ABC Tubulinopathy.

Tubulinopathies are a group of recently described diseases characterized by mutations in the tubulin genes. Mutations in TUBB4A produce diseases such as dystonia type 4 (DYT4) and hypomyelination with atrophy of the basal ganglia and cerebellum (H-ABC), which are clinically diagnosed by magnetic resonance imaging (MRI). We propose the taiep rat as the first animal model for tubulinopathies. The spontaneous mutant suffers from a syndrome related to a central leukodystrophy and characterized by tremor, ataxia, immobility, epilepsy, and paralysis. The pathological signs presented by these rats and the morphological changes we found by our longitudinal MRI study are similar to those of patients with mutations in TUBB4A. The diffuse atrophy we found in brain, cerebellum and spinal cord is related to the changes detectable in many human tubulinopathies and in particular in H-ABC patients, where myelin degeneration at the level of putamen and cerebellum is a clinical trademark of the disease. We performed Tubb4a exon analysis to corroborate the genetic defect and formulated hypotheses about the effect of amino acid 302 change on protein physiology. Optical microscopy of taiep rat cerebella and spinal cord confirmed the optical density loss in white matter associated with myelin loss, despite the persistence of neural fibers.

Liquid refractive index measured through a refractometer based on diffraction gratings.

We developed two versions of refractometers to measure the refractive index of liquids. One refractometer comprises a glass cell with a surface relief grating on the inner face of one of its walls, while the other one is a microfluidic channel in the form of serpentine that behaves as a grating. Measurements of the liquid refractive index were performed by sensing the first order intensity. Several liquids have been used including an organic one. Calibration plots are shown.

3,3'-thiodipropanol as a versatile refractive index-matching mounting medium for fluorescence microscopy.

High resolution fluorescence microscopy requires optimization of the protocols for biological sample preparation. The optical and chemical characteristics of mounting media are among the things that could be modified to achieve optimal image formation. In our search for chemical substances that could perform as mounting media, 3,3'-thiodipropanol (TDP) emerged as a sulfide with potentially interesting characteristics. In this work, several tests of its performance as a mounting medium for fluorescence microscopy of biological samples were performed, including the labeling of filamentous actin with fluorescent phalloidins. The refractive index dispersion curve of pH-adjusted TDP was experimentally obtained in the visible range and compared to the dispersion curves of commercial and lab-made mounting media. The effects on the fluorescence of commonly used dyes were tested by using TDP as a solvent and measuring the relative fluorescence quantum yield of the dyes. By being able to mix TDP in any concentration with water and 2,2'-thiodiethanol (TDE), it was possible not only to fine-tune the refractive index of the resulting solution, but also to preserve the compatibility of TDP with the most popular and efficient fluorescent actin staining used in biological microscopy.

Stimuli-Responsive Systems in Optical Humidity-Detection Devices.

The use of electronic devices to measure Relative Humidity (RH) is widespread. However, under certain circumstances, for example when explosive gases are present, a spark-free method should be used. Here we suggest the use of stimuli-responsive materials, like gelatin and interpenetrated polymers, to detect RH with an optical method. These materials are hydrophilic. When water vapor is absorbed by the films the molecules attach to the films molecular network. The result is that the film thickness increases and their refractive index changes. To detect the change of these two parameters an optical method based on diffraction gratings is employed. Surface diffraction gratings are recorded on the films. Then gratings are placed in an optical configuration that is immersed in a climatic chamber. A light beam is sent to the grating where it is diffracted. Several light orders appear. Due to the absorption of water molecules the films swell and grating surface modulation changes. This implies that the diffracted orders intensity changes. A calibrating plot relating intensity as a function of RH is obtained.

Different routes into the glass state for soft thermo-sensitive colloids.

We report an experimental and theoretical investigation of glass formation in soft thermo-sensitive colloids following two different routes: a gradual increase of the particle number density at constant temperature and an increase of the radius in a fixed volume at constant particle number density. Confocal microscopy experiments and the non-equilibrium self-consistent generalized Langevin equation (NE-SCGLE) theory consistently show that the two routes lead to a dynamically comparable state at sufficiently long aging times. However, experiments reveal the presence of moderate but persistent structural differences. Successive cycles of radius decrease and increase lead instead to a reproducible glass state, indicating a suitable route to obtain rejuvenation without using shear fields.

An immunoconjugated up-conversion nanocomplex for selective imaging and photodynamic therapy against HER2-positive breast cancer.

Photodynamic therapy represents a very attractive therapeutic tool considered to be effective, minimally invasive and minimally toxic. However, conventional photodynamic therapy actually has two main constraints: the limited penetration depth of visible light needed for its activation, and the lack of selectivity. Considering this, this work reports the synthesis and evaluation of a novel nanoconjugate for imaging and selective photodynamic therapy against HER2-positive breast cancer, a particularly aggressive form of the disease. It was demonstrated that upon 975 nm near infrared light exposure, the red emission of the NaYF4:Yb,Er up-conversion nanoparticles (UCNPs) can be used for optical imaging and simultaneously represent the source for the excitation of a covalently bound zinc tetracarboxyphenoxy phthalocyanine (ZnPc), a photosensitizer that in turn transfers energy to ground state molecular oxygen to produce cytotoxic singlet oxygen. The specificity of our nanoconjugates was achieved by immunoconjugation with Trastuzumab (Tras), a specific monoclonal antibody for selective detection and treatment of HER2-overexpressing malignant breast cancer cells. Selective tracking of SKBR-3 HER2-positive cells was verified by confocal microscopy analysis, and the photodynamic therapy effect was considerably improved when Trastuzumab was incorporated into the nanoconjugate, the UCNPs-ZnPc-Tras being practically inert in the absence of infrared light exposure but reducing the HER2-positive cell viability up to 21% upon 5 min of the irradiation. This theranostic nanoconjugate represents a valuable alternative for HER2-positive breast cancer imaging and selective photodynamic therapy.

Interaction of TGA@CdTe Quantum Dots with an Extracellular Matrix of Haematococcus pluvialis Microalgae Detected Using Surface-Enhanced Raman Spectroscopy (SERS).

The present study reports the localization and interaction of thioglycolic acid (TGA) capped CdTe quantum dots (TGA@CdTe QDs) within the extracellular matrix (ECM) of Haematococcus pluvialis (Chlorophyceae) microalgae (HPM) after an incubation period of 5 min. Changes in the Raman spectrum of HPM induced by the adsorption of the TGA@CdTe QDs are successfully found by using naked gold anisotropic structures as nano-sensors for surface-enhanced Raman scattering (SERS effect). Raman spectroscopy results show that TGA@CdTe QDs interact with the biomolecules present in the ECM. Sample preparation and characterization by complementary techniques such as confocal and electron microscopy are also used to confirm the presence and localization of the nanoparticles in the algae. This research shows new evidence on early accumulation of QDs in plant cells and would further improve our understanding about their environmental impact.

Revealing the structure of stereociliary actin by X-ray nanoimaging.

Hair cell stereocilia are crucial for hearing and the sense of balance. They include an array of accurately packed, parallel actin filaments and act as levers, which transform mechanical deformation into neuronal signals. The length of vestibular stereocilia reaches several micrometers, whereas, for individual microfilaments, the diameter and therefore the characteristic length scale in the lateral direction is on the order of a few nanometers. These orders of magnitude render X-rays an ideal tool for investigating actin packing, and numerous studies on reconstituted in vitro systems have revealed important information. Here we report on the characterization of intact stereocilia using two nanoscale X-ray techniques. We use X-ray ptychography to image stereocilia with quantitative phase contrast and high dose efficiency, showing stereocilia with diameters and lengths in the expected range. We further employ X-ray nanodiffraction using a nanofocused X-ray beam on the same order of magnitude as the width of a stereocilium. Despite the small probe volume we can clearly visualize the stereocilia bundles. From the individual diffraction patterns we determine the local orientation of the actin structures and can clearly correlate them with the corresponding visible-light fluorescence images. Furthermore, azimuthal integration of individual diffraction patterns reveals distinct intensity curves, showing modulations of the signal, which reflect the relevant length scales and pronounced order in the biological system. The applied techniques are not limited to the studies on stereocilia but have the potential of being applied to many biological and soft-matter systems, in particular if a pronounced degree of order is present.

Multi-isotope imaging mass spectrometry reveals slow protein turnover in hair-cell stereocilia.

Hair cells of the inner ear are not normally replaced during an animal's life, and must continually renew components of their various organelles. Among these are the stereocilia, each with a core of several hundred actin filaments that arise from their apical surfaces and that bear the mechanotransduction apparatus at their tips. Actin turnover in stereocilia has previously been studied by transfecting neonatal rat hair cells in culture with a ß-actin-GFP fusion, and evidence was found that actin is replaced, from the top down, in 2-3 days. Overexpression of the actin-binding protein espin causes elongation of stereocilia within 12-24 hours, also suggesting rapid regulation of stereocilia lengths. Similarly, the mechanosensory 'tip links' are replaced in 5-10 hours after cleavage in chicken and mammalian hair cells. In contrast, turnover in chick stereocilia in vivo is much slower. It might be that only certain components of stereocilia turn over quickly, that rapid turnover occurs only in neonatal animals, only in culture, or only in response to a challenge like breakage or actin overexpression. Here we quantify protein turnover by feeding animals with a (15)N-labelled precursor amino acid and using multi-isotope imaging mass spectrometry to measure appearance of new protein. Surprisingly, in adult frogs and mice and in neonatal mice, in vivo and in vitro, the stereocilia were remarkably stable, incorporating newly synthesized protein at <10% per day. Only stereocilia tips had rapid turnover and no treadmilling was observed. Other methods confirmed this: in hair cells expressing ß-actin-GFP we bleached fiducial lines across hair bundles, but they did not move in 6 days. When we stopped expression of ß- or γ-actin with tamoxifen-inducible recombination, neither actin isoform left the stereocilia, except at the tips. Thus, rapid turnover in stereocilia occurs only at the tips and not by a treadmilling process.

ATP-mediated cell-cell signaling in the organ of Corti: the role of connexin channels.

UNLABELLED: Connexin 26 (Cx26) and connexin 30 (Cx30) form hemichannels that release ATP from the endolymphatic surface of cochlear supporting and epithelial cells and also form gap junction (GJ) channels that allow the concomitant intercellular diffusion of Ca(2+) mobilizing second messengers. Released ATP in turn activates G-protein coupled P2Y(2) and P2Y(4) receptors, PLC-dependent generation of IP(3), release of Ca(2+) from intracellular stores, instigating the regenerative propagation of intercellular Ca(2+) signals (ICS). The range of ICS propagation is sensitive to the concentration of extracellular divalent cations and activity of ectonucleotidases. Here, the expression patterns of Cx26 and Cx30 were characterized in postnatal cochlear tissues obtained from mice aged between P5 and P6. The expression gradient along the longitudinal axis of the cochlea, decreasing from the basal to the apical cochlear turn (CT), was more pronounced in outer sulcus (OS) cells than in inner sulcus (IS) cells. GJ-mediated dye coupling was maximal in OS cells of the basal CT, inhibited by the nonselective connexin channel blocker carbenoxolone (CBX) and absent in hair cells. Photostimulating OS cells with caged inositol (3,4,5) tri-phosphate (IP(3)) resulted in transfer of ICS in the lateral direction, from OS cells to IS cells across the hair cell region (HCR) of medial and basal CTs. ICS transfer in the opposite (medial) direction, from IS cells photostimulated with caged IP(3) to OS cells, occurred mostly in the basal CT. In addition, OS cells displayed impressive rhythmic activity with oscillations of cytosolic free Ca(2+) concentration ([Ca(2+)](i)) coordinated by the propagation of Ca(2+) wavefronts sweeping repeatedly through the same tissue area along the coiling axis of the cochlea. Oscillations evoked by uncaging IP(3) or by applying ATP differed greatly, by as much as one order of magnitude, in frequency and waveform rise time. ICS evoked by direct application of ATP propagated along convoluted cellular paths in the OS, which often branched and changed dynamically over time. Potential implications of these findings are discussed in the context of developmental regulation and cochlear pathophysiology. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (doi:10.1007/s11302-010-9192-9) contains supplementary material, which is available to authorized users.

Purinergic signalling and intercellular Ca2+ wave propagation in the organ of Corti.

Extracellular ATP is a key neuromodulator of visual and auditory sensory epithelia. In the rat cochlea, pharmacological dissection indicates that ATP, acting through a highly sensitive purinergic/IP(3)-mediated signaling pathway with (little or) no involvement of ryanodine receptors, is the principal paracrine mediator implicated in the propagation of calcium waves through supporting and epithelial cells. Measurement of sensitivity to UTP and other purinergic agonists implicate P2Y(2) and P2Y(4) as the main P2Y receptor isoforms involved in these responses. Ca2+ waves, elicited under highly reproducible conditions by carefully controlling dose (1 microM) and timing of focal agonist application (0.2s), extended over radial distance greater than 160 microm from the source, identical to those activated by damaging single outer hair cells. Altogether, these results indicate that intercellular calcium waves are a robust phenomenon that confers a significant ability for cell-cell communication in the mammalian cochlea. Further ongoing research will reveal the roles that such Ca2+ waves play in the inner ear.